The present invention relates to methods for identifying and producing L-nucleic acids that interact with a target molecule having a natural configuration, as well as to the L-nucleic acids produced by means of this method. Furthermore, the invention relates to the use of the D-nucleic acids binding to the optical antipode of the target molecule as a matrix for producing L-nucleic acids with an identical sequence, as well as to pharmaceutical compositions, kits, diagnostic agents and sensor systems containing the L-nucleic acids of the invention.
In the past few years, new technologies have been established in order to use nucleic acids in a manner previously unanticipated. Among these are, e.g., the use of such molecules as catalysts, inhibitors or stimulators of biochemical reactions that take place within or outside of the cell. There is hardly any doubt that these technologies will in the future play a dominant role in the fields of medicine, pharmaceutical diagnostics, biotechnology and agriculture.
An essential part of some of the new DNA and RNA techniques is in vitro selection or evolution (cf. for example the review article of J. W. Szostak, TIBS 17 (1992), 89 to 93, Famulok and Szostak, Angew. Chemie 104 (1992), 1001-1011 and Gold et al., Annu. Rev. Biochem. (1995)). These techniques are based on the working methods of biological systems. Thereby, novel DNA and RNA molecules with desired properties may be obtained from a combinatorial library of heterogeneous nucleic molecules by means of variation, selection and replication. Thus, such an in vitro system contains all factors present in biological evolution. It may rightfully be referred to as an in vitro evolution which many times accelerates the speed of natural methods. As long as no additional variation steps occur in this system, it is no in vitro evolution, but merely an in vitro selection method.
From a group of up to 1018 different RNA species, the RNA molecules with highly affine binding properties or catalytic properties may be isolated by means of a cyclic process of the polymerase chain reaction (PCR), transcription, selective binding and reverse transcription; cf. Gold et al., loc. cit. One of the essential advantages of this method is the fact that it is not necessary to know the structure of the molecule to be selected; molecules with the xe2x80x9ccorrectxe2x80x9d structure are filtered out from the starting population by means of a selection step and, if desired, they may subsequently be sequenced. The principle of this selection or evolution process is schematically depicted in FIG. 1.
Examples for such in vitro selection or evolution methods have been provided by Tuerk and Gold, Science 249 (1990), 505-510, Berzal-Herranz et al., Genes and Development 6 (1992), 129-134 and by Robertson and Joyce, Nature 344 (1990), 467-468. These work groups successfully selected functional ribonucleic acids cleaving or binding a predetermined nucleic acid differing from the substrate by means of slightly varying experimental approaches. In a more recent study, Lehmann and Joyce have shown that a ribozyme""s metal ion specificity may be changed from Mg2+ to Ca2+ by such an in vitro evolution process (Nature 361 (1993), 182-185).
Highly affine RNA, but also DNA, molecules may not only be constructed for the purpose of interaction with other nucleic acids, but primarily for their interacting with proteins, with other, smaller molecules of the cell or with synthetic compounds. Attempts may also be made for interactions with the cellular receptors or with viral particles. Usually, such interactions of highly affine nucleic acids have the purpose of inhibiting or stimulating a biological function or of prompting a signal in sensor systems.
The advantage of nucleic acid libraries when compared to combinatorial libraries of other oligomers or polymers may be found in the dual nature of nucleic acids. The molecules possess a genotype (a sequence capable of propagation) and a phenotype (a functional structure). This enables the amplification of functional molecules from very large combinatorial libraries and their identification by means of sequencing. The additional labeling of the molecule library in order to identify functional variants e.g. by xe2x80x9ctaggingxe2x80x9d (Janda, Proc. Natl. Acad. Sci. USA 91 (1994), 10779-10785) and the technical problems associated therewith (Gold et al., loc. cit.; Gold, J. Biol. Chem. 270 (1995), 13581-13584) may be avoided. By using combinatorial phage libraries in order to identify peptide motifs (Scott and Smith, Science 249 (1990), 386-390, Devlin et al., Proc. Natl. Acad. Sci. USA (1990), 6378-6382), which also involves the combination of genotype and phenotype, other disadvantages occur. Whereas oligonucleotides with only 25 nucleotides may already form very stable structures, comparable oligopeptides possess large conformational liberties (Gold et al., loc. cit.). The structural liberty of peptides and the entropic disadvantages in the interaction with target structures resulting therefrom limit the possibilities of using peptides, as long as high affinities and specificities are necessary for the application of the molecules. This limitations also occur in the selection of biologically stable D-peptides by means of phage libraries (Schumacher et al., Science 271 (1996); 1854-1857). The use of cyclic peptides may not offset these basic disadvantages (Gold et al., loc. cit.).
The particular disadvantage in using combinatorial nucleic acid libraries instead of other oligomers or polymers is the low stability of nucleic acids in biological liquids.
However, all selection and evolution processes known so far are only capable of producing highly affine or catalytic RNAs or DNAs in natural form, i.e. with D-ribose or D-deoxyribose as a basic component. During use in a biological environment these molecules are degraded by enzymes. The degradation leads to a short term of effect of these highly affine or catalytic nucleic acids.
Although it is possible after the selection of unmodified nucleic acids to introduce a targeted modification in order to slow down the enzymatic degradation, the influence of this modification on the structure and thereby on the functionality of the nucleic acids may, however, not be predicted. Furthermore, altered, undesired properties cannot be anticipated. In addition, the degradation of chemically modified DNAs or RNAs leads to products that may influence the cell metabolism in a serious and disadvantageous manner in the form of analogues of nucleosides, nucleotides or oligonucleotides.
It is furthermore possible to integrate into the process modified nucleoside triphosphates which increase the stability of the nucleic acids. Examples for this procedure have been described by Jellinek et al., Biochemistry 34 (1995), 11363-11372. and Eaton and Pieken, Annu. Rev. Biochem. 64 (1995), 837-863. As the nucleoside triphosphates must be compatible to the polymerases used, the range of possible modifications is very limited. Furthermore, it has to be expected that the degradation of these modified nucleic acids leads to particularly toxic effects.
Thus, the technical problem underlying the present invention was to provide processes, by means of which highly affine nucleic acid molecules can be produced via in vitro selection or evolution, which do not exhibit the above-described disadvantages mentioned in the prior art. This technical problem is solved by the embodiments characterized in the claims. Thus, the present invention relates to a process for producing L-nucleic acids interacting with a target molecule having natural configuration, said process comprising the following steps:
(a) producing a heterogeneous population of D-nucleic acids;
(b) bringing the population mentioned in step (a) into contact with the optical antipode of the target molecule;
(c) separating the D-nucleic acids interacting with the optical antipode of the target molecule;
(d) sequencing the D-nucleic acids interacting with the optical antipode of the target molecule;
(e) synthesizing L-nucleic acids, the sequences of which are identical with the sequences of the D-nucleic acids determined in step (d).
In this context, an xe2x80x9cL-nucleic acidxe2x80x9d may be any nucleic acid occurring in the naturally not occurring L-configuration. This means that instead of an D-ribose or D-deoxyribose, which naturally form the backbone of a nucleic acid, L-ribose or L-deoxyribose are used as the basic component of the L-nucleic acids. The term xe2x80x9ca target molecule having natural configurationxe2x80x9d may comprise any molecule capable of binding a nucleic acid, as long as it occurs in its natural structure. Examples for such molecules are proteins composed of L-amino acids, L-amino acids, nucleic acids consisting of D-nucleotides, as well as D-sugars and more complex sugar molecules consisting thereof.
The production of a heterogeneous D-nucleic acid population may be carried out by any means of processes known in the prior art. Examples for such processes are the amplification of genomic fragments (Kinzler and Vogelstein., Nucl. Acids Res. 17 (1989), 3645-3635) or the chemical solid phase synthesis of DNA molecules by means of synthesizers (Thiesen and Bach, Nucl. Acids Res. 18 (1990), 3203-3209 and Pollock and Treisman, Nucleic Acids Res. 18 (1990), 6197-6204). Moreover, the skilled person is capable of varying these processes, depending on the experimental arrangement, which also lead to the desired results. The nucleic acids may consist of a desired number of D-nucleotides. The D-nucleotides preferably exhibit arbitrary (not previously determined) nucleotides in at least 15 positions.
The heterogeneous D-nucleic acid population exhibits any desired number of members, preferably of at least 109 members.
In step (b), the D-nucleic acids are brought into contact with the optical antipode of the target molecule, allowing for the interaction of the (highly) affine nucleic acid with the optical antipode of the target molecule.
In this context, the term xe2x80x9coptical antipode of the target moleculexe2x80x9d means the enantiomeric form of a (macro)molecule occurring in natural configuration. The optical antipode of the target molecule can be produced according to methods described in the prior art. Thus, for example Orata et al. (Nucl. Acids Res. 20 (1992), 3325 to 3332) have described the synthesis of a hexadeoxyribonucleotide consisting of L-deoxyriboses. Furthermore, the L-nucleic acids may be produced as described in Example 1. The optical antipode of a L-(poly)peptide may for example be produced according to the processes described by Milton et al. (Science 258 (1992), 1445-1448) or Muir (Structure 3 (1995), 649-652).
The separation of D-nucleic acids not interacting with the target molecule takes place according to processes known from the prior art. The separation may be carried out, for example, by means of a column-chromatographic process, whereby the optical antipode of the target molecule is bound to the column material and affine or, as the case may be, highly affine D-nucleic acids are held back under suitable conditions. The nucleic acids bound in such a way can be eluted from the column material after the washing of the unbound nucleic acids. However, the separation may also be carried out by means of separating techniques such as filtering methods or magnetic particles. Moreover, the skilled person is capable of modifying the processes described in the prior art for its own special needs.
After separating the non-interacting nucleic acids from the interacting nucleic acids, the interacting nucleic acids are separated from the optical antipode of the target molecule. Provided that a limited heterogeneity of the population of step (a) had existed and that it may be assumed that a sufficient number of identical or similar molecules occurs, the D-nucleic acids previously interacting with the optical antipode of the target molecule can be directly sequenced. Suitable sequencing techniques are known from the prior art; cf. Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd edition, 1989, Cold Spring Harbor Laboratory, Cold Spring Harbor. Provided that in this selection technique nucleic acids with different sequences bind to the optical antipode of the target molecule and are subsequently sequenced, a sequence information may be obtained which is ambiguous at various positions. However, it may be taken into consideration that a number of nucleic acids are conserved at certain positions. This shows their role in the binding of the optical antipode of the target molecule, as was shown by Blackwell et al. (Blackwell and Weintraub, Science 250 (1990), 1104 to 1110, Blackwell et al., Science 250 (1990), 1149 to 1151). This limited information already enables the skilled person to synthesize an L-nucleic acid capable of binding to the desired target molecule.
For many applications, the process of identifying mirror-symmetrical nucleic acids is rather expansive since at first the antipode of the target structure has to be prepared in enantiomerically pure form. The racemat cleaving may be circumvented if the enantiomerically pure target structure is at disposal. In another preferred embodiment the invention therefore relates to a process that causes the interaction of the D-nucleic acids with the racemic mixture of a target molecule. The separation of the D-nucleic acid not interacting with the racemic mixture of the target molecule is carried out according to methods known in the art. The separation may for example take place by means of a column-chromatographic method, whereby the racemic mixture of the target molecule is bound to the column material, and affine or, as the case may be, highly affine D-nucleic acids are held back under suitable conditions.
Hereby, nucleic acids, which may interact (a) with the target structure, (b) the mirror-symmetrical target structure or (c) with both isomers due to the low chiral discrimination, bind at first. The share of highly affine D-nucleic acids bound to the naturally occurring enantiomer and possessing a low chiral specificity may be washed with the natural target molecule by means of elution. The remaining D-nucleic acids bound to the optical antipode may subsequently be specifically eluted with the racemic mixture. Thereby, it is possible to carry out the process of mirror-symmetrical selection or evolution even without isolating the optical antipode of the target molecule.
The synthesis of the L-nucleic acids is carried out according to processes known from the prior art (Urata et al., loc. cit.) or according to the method described in Example 1.
Considering L. Pasteur""s studies (e.g. in Soc. Chim. Paris 1860, 1, (1860)) with regard to enantiomers, it may be postulated that the naturally occurring nucleic acids possess optical antipodes the basic component of which is L-ribose or L-deoxyribose. In accordance with the present invention""s object, the chemical solid phase synthesis of L-RNA and L-DNA have now successfully been established in any desired sequence (cf. Example 1).
On examining a nucleic acid selected according to the method of the invention, it may be postulated that a corresponding L-polymer of identical sequence interacts with the optical antipode of the target molecule in the same manner. If the enantiomer of the target molecules is utilized in the evolution or selection process, sequence information may be obtained allowing for the production of highly affine or catalytic L-RNAs or L-DNAs. The principle of this method is depicted in FIG. 2. The advantage of this method referred to as mirror-symmetrical selection or evolution is the high stability of the products in biological environment. By means of this method, an L-oligoribonucleotide was for the first time identified, which binds to the naturally occurring D-adenosine with high affinity (cf. Example 2). After incubating the L-oligoribonucleotide in human serum, no degradation could be stated. In subsequent investigations it could be proved that L-oligoribonucleotide only binds D-adenosine and that D-oligoribonucleotide only binds L-adenosine. This shows that D- and L-oligonucleotides with identical, covalently linked nucleotide bonds fold up in the same manner in order to acquire three-dimensional structure, and that the nucleotide sequence only determines the tertiary structure of a nucleic acid. The affinity of L-RNA to D-adenosine described in Example 2 (dissociation factor 1.8 xcexcM) lies within the range of the affinities of D-RNA to nucleosides achieved so far by means of in vitro evolution (cf. Connell and Yarus, Science 264 (1994), 1137-1141 and Huizenga and Szostak, Biochemistry 34 (1995), 656-665). Thus, it has been shown for the first time that L-nucleic acids are capable of developing equally high affinities to naturally occurring chiral target substances as D-nucleic acids. The principle of the process could be confirmed by the identification of arginine-specific ligands (cf. Example 3).
The mirror-symmetrical evolution or selection exhibits the particular feature that selection takes place with regard to the optical antipode of the target molecule. This particularity has two essential advantages: on the one hand, the optical antipode of the target molecule is not so easily degraded during the process of the invention, as is the case with naturally occurring enantiomers. This feature plays an important role in the case of the target molecule being an RNA. On the other hand, the herein-described method in particular offers an advantage unknown in the prior art, namely, that after the finished selection or evolution procedure the enantiomer of the selected (highly) affine D-nucleic acid is produced.
This L-nucleic acid not only exhibits the above-described high stability in a biological environment which grants its long-termed effect in biological systems. As a consequence of this stability, which is due to the lack of suitable degrading enzymes in biological systems, metabolites of these nucleic acids which might represent a sanitary hazard are no longer formed. Moreover, it is likely that these L-nucleic acids are not or only to small extent dealt with by the immune system and therefore only prompt a slight immune reaction, if any. The products of the process of the invention may therefore be used as pharmaceutical compositions without further hesitation.
Thus, the process of the invention provides products, which are mirror-symmetrical reflections of naturally occurring nucleic acids. These products have a defined sequence and possess a high binding affinity against a given ligand or catalyze a desired reaction at a desired target molecule. Thereby, chemically synthesized polymers are provided which exhibit a high biological stability and may be used analogously to monoclonal antibodies. The versatile hydrolytic properties, but possibly also synthetic properties, render the molecules of the invention interesting for chemical or pharmaceutical uses.
In a further preferred embodiment the invention relates to a process, wherein the following step is additionally added subsequently to step (c):
(ca) amplifying the D-nucleic acids interacting with the optical antipode of the target molecule.
By means of this embodiment of the invention, in particular D-nucleic acids may be selected which are present in the heterogeneous starting population in low numbers. The principle of this preferred embodiment is depicted in FIG. 3. The additional amplification step increases the (highly) affine D-nucleic acids obtained by binding to the optical antipode of the target molecule. Said D-nucleic acids may be further enriched in a repeated selection step or they may be directly sequenced after amplification. Amplifications may, however, also be carried out in isothermal systems. Such systems have e.g. been described by Guatelli et al. (Proc. Natl. Acad. Sci. USA 87 (1990), 1874-1878), and Walker et al. (Nucl. Acids Res. 20 (1992), 1691-1696).
In another preferred embodiment of the process of the invention the D-nucleic acids of the step (a) population exhibit primer binding sites or, as the case may be, complementary sequences to primer binding sites at their 5xe2x80x2 and 3xe2x80x2 ends, which allow for an amplification by PCR of the D-nucleic acids obtained in step (ca).
This embodiment of the process of the invention is particularly suitable if the D-nucleic acid is selected on an RNA level. Moreover, use can be made of transcription by means of a DNA-dependent RNA polymerase as an additional amplification step apart from PCR. A combination of both these amplification steps thus enables a particularly high yield of D-nucleic acid material interacting with the optical antipode of the target molecule.
In a further preferred embodiment of the process of the invention the following step is added subsequently to step (ca):
(cb) bringing the amplified D-nucleic acids into contact with the optical antipode of the target molecule.
This step is followed by step (b) and possibly (ca) before carrying out step (d), whereby the steps (cb), (b) and possibly (ca) may be repeated in this order once or several times.
The amplification of the D-nucleic acids isolated in the selection steps via PCR leads to the enrichment of the desired D-nucleic acids in a comfortable manner. In a preferred embodiment of the process of the invention, in which several cycles of bringing the amplified D-nucleic acids into contact with the optical antipode of the target molecule, subsequent separating of the unbound molecule (selection) and amplification take place, finally the nucleic acids with the highest binding affinities are selected. By varying the molar ratio of the optical antipode of the target molecule and D-nucleic acids, selection may take place with respect to a majority of (highly) affine D-nucleic acids (ratio  greater than 1) or to one or only a few nucleic acids (ratio xe2x89xa61). Correspondingly, step (e) results in one or a few highly affine L-nucleic acids or in a majority of (highly) affine L-nucleic acids. The respective results may be achieved by varying the number of selection steps, whereby a high number of selection steps will finally result in one or in a few D-nucleic acids with high binding affinities.
The amplification step of the process of the invention may comfortably be carried out by means of PCR. PCR is a well-established method in the prior art, the principles of which have been described in Sambrook, loc. cit. RNA as well as DNA may be amplified via PCR, whereby a process step resulting in the translation of RNA into a corresponding cDNA by means of reverse transcriptase may be integrated into the amplification of RNA.
However, the amplification may also be achieved by means of other techniques known from the prior art.
In another preferred embodiment of the process of the invention nucleotides are integrated during amplification into the nucleotide strands to be synthesized anew.
These nucleotides do not occur at the nucleotide position of the D-nucleic acids interacting with the target molecule and mentioned in step (a).
By this preferred embodiment selection may take place with regard to novel D-nucleic acid species not occurring in the starting population in a similar manner as in the biological in vivo evolution. Thus, this is a case of in vitro evolution. Several variants of this process are conceivable, all falling under the scope of protection of the present invention.
On the one hand, amplification as such may be carried out via a process exhibiting a certain rate of error during the integration of the nucleotides in the strand to be synthesized anew. PCR is a known process of that kind.
Provided that the binding site necessary for optimal binding exhibits more than about 25 nucleotides, there is a certain probability that the corresponding sequence is not contained in the original population of step (a). This is due to the fact that, starting from a certain length of the nucleotide sequence (laying within the range of 25 nucleotides) not any possible sequence may be contained in this population for reasons of practicability.
In case that the optimal sequence for binding to the antipode of the target molecule is indeed not present within the population of step (a), it may, however, be selected with the process of the invention. This is carried out by first isolating a D-nucleic acid with suboptimal binding affinity from the population (within the population, however, this sequence is the molecule with optimal binding affinity). Subsequently, this sequence is mutagenized during amplification. Such mutagenesis techniques are known from the prior art (cf. for example Light and Lerner, Bioorg. Med. Chem. 3 (1995), 995-967 and Pannekoek et al., Gene 128 (1993), 135-140). Thereby, the complete sequence may be mutagenized. The mutagenized sequence is subjected to further selection steps whereby several amplification, mutagenesis and subsequently selection cycles follow each other until a D-nucleic acid with optimal binding properties has been obtained.
Furthermore, for practical reasons, a short D-nucleic acid exhibiting the optimal binding properties of its population may at first be incorporated. The positions essential for binding are determined by means of methods known from the prior art, and the other nucleotide areas are substituted by longer sections. This procedure is again followed by one or more amplification and selection cycles. The parts of the sequence of the thus-determined molecule with optimal binding properties which are unessential for binding are again substituted by a randomized sequence. This process step has for example been described in W 91/19813 in another context and was there designated as xe2x80x9cwalkingxe2x80x9d.
In a further embodiment of the present invention L-nucleic acids are made to interact directly with a target molecule having natural configuration. Thus, the invention relates to a method for producing L-nucleic acids comprising the following steps:
(a) producing a heterogeneous population of L-nucleic acids;
(b) bringing the population of step (a) into contact with the target molecule;
(c) separating the L-nucleic acids not interacting with the target molecule;
(d) sequencing the L-nucleic acids interacting with the target molecule;
(e) synthesizing L-nucleic acids the sequence of which is identical to the sequences determined in step (d).
The production of a heterogeneous population of L-nucleic acids takes place according to methods known from the prior art (Urata et al., loc. cit.) or according to the method described in Example 1. After separating the non-interacting L-nucleic acids from the interacting L-nucleic acids, the interacting nucleic acids are separated from the target molecule. The L-nucleic acids are then iteratively singled out according to the method set forth in Example 4. Instead of the L-proteins used in Example 4, D-proteins are used in this process. The optical antipodes of the enzymes may be produced according to methods described by Milton et al. (loc. cit.) or by Muir (loc. cit.). The strands may be separated by means of the method described in Example 4 or by means of any method described in the prior art. Examples for such methods are strand separation gels (Maxam and Gilbert, Proc. Natl. Acad. Sci. 78 (1977), 560-564; Maniatis, loc. cit.) or solid phase sequencing (Hultman et al., Nucl. Acids Res. 17 (1989), 4937-4946, Hultman et al., BioTechniques 10 (1991), 84-93). A further possibility of obtaining a single-stranded DNA after PCR is to utilize a primer with an internal spacer (e.g. polyethylene glycol) (Williams and Bartel, Nucl. Acids Res. 23, (1995), 4220-4221). The L-deoxynucleoside triphosphates and L-dideoxynucleoside triphosphate necessary for sequencing are obtained by chemically synthesizing the L-nucleosides described in Example 1 according to methods described in the prior art for D-nucleosides. In the synthesis of triphosphate, the four L-deoxynucleosides are first phosphorylized at the 5xe2x80x2-position (Yoshikawa et al., Tetrahedron Lett. (50), 1967, 5065-5068). The 5xe2x80x2-monophosphates are then transformed into their 5xe2x80x2-triphosphates (Hoard and Ott, J. Am. Chem. Soc. 87 (1965), 1785-1788). In order to synthesize the L-dideoxynucleotide triphosphates, the L-deoxynucleosides are at first transformed into L-dideoxynucleosides. The synthesis of the pyrimidine-L-dideoxynucleosides may be carried out in a multi-step process according to Horwitz et al. (J. Org. Chem. 32 (1967), 817-818) and Joshi et al. (J. Chem. Soc. (1992), 2537-2544). The guanosine-L-dideoxynucleoside may be synthesized according to Herdewijn et al. (J. Med. Chem. 31 (1988), 2040-2048), and the adenosine-L-dideoxynucleoside may be synthesized according to Chu et al. (J. Org. Chem. 54 (1989), 2217-2225). The four L-dideoxynucleosides obtained thereby are then converted into their respective 5xe2x80x2-triphosphates via the intermediate step of 5xe2x80x2-monophosphates by means of the above-mentioned methods of Yoshikawa et al. (Tetrahedron Lett. 50 (1967), 5065-5068) and Hoard and Ott (Hoard and Ott, J. Am. Chem. Soc. 87 (1965), 1785-1788). In a further preferred embodiment the invention relates to a process whereby the following step is additionally introduced after step (c):
(ca) amplifying the L-nucleic acids interacting with the target molecule.
The propagation of the L-nucleic acids is achieved by means of D-polymerases produced according to the methods described by Milton et al. (loc. cit.) or Muir (loc. cit.). Particularly such L-nucleic acids which are present in low amounts in the heterologous starting population may be selected by this embodiment of the method of the invention. The principle of this preferred embodiment is depicted in FIG. 5. The additional amplification step increases the number of (highly) affine L-nucleic acids obtained by binding to the target molecule. These may be enriched by a repeated selection step or sequenced after amplification.
The amplifications, however, may also be carried out in isothermal systems with the help of corresponding D-polymerases. Such systems have been described for L-polymerases for example by Guatelli et al. (loc. cit.) and Walker et al. (loc. cit.).
In another preferred embodiment of the process of the invention the L-nucleic acids of the step (a) population exhibit primer binding sites or, as the case may be, complementary sequences to primer binding sites at their 5xe2x80x2 and 3xe2x80x2 ends, which allow for an amplification of the L-nucleic acids obtained in step (ca) obtained by mirror-symmetrical PCR.
This embodiment of the process of the invention is particularly suitable if the L-nucleic acid is selected on an RNA level. Moreover, use can be made of mirror-symmetrical transcription by means of a DNA-dependent D-RNA polymerase as an additional amplification step apart from mirror-symmetrical PCR. A combination of both these amplification steps thus enables a particularly high yield of L-nucleic acid material interacting with the optical antipode of the target molecule.
In a further preferred embodiment of the process of the invention the following step is added subsequently to step (ca):
(cb) bringing the amplified L-nucleic acids into contact with the target molecule.
This step is followed by step (b) and possibly (ca) before carrying out step (d), whereby the steps (cb), (b) and possibly (ca) may be repeated in this order once or several times.
The amplification of the L-nucleic acids isolated in the selection steps via mirror-symmetrical PCR leads to the enrichment of the desired L-nucleic acids in a comfortable manner. In a preferred embodiment of the process of the invention, in which several cycles of bringing the amplified L-nucleic acids into contact with the target molecule, subsequent separating of the unbound molecule (selection) and amplification take place, finally the nucleic acids with the highest binding affinities are selected. By varying the molar ratio of the target molecule and L-nucleic acids, selection may take place with respect to a majority of (highly) affine L-nucleic acids (ratio  greater than 1) or to one or only a few nucleic acids (ratio xe2x89xa61). Correspondingly, step (e) results in one or a few highly affine L-nucleic acids or in a majority of (highly) affine L-nucleic acids. The respective results may be achieved by varying the number of selection steps, whereby a high number of selection steps will finally result in one or in a few L-nucleic acids with high binding affinities.
In another preferred embodiment of the process of the invention nucleotides are integrated during amplification into the nucleotide strands to be synthesized anew. These nucleotides do not occur at the nucleotide position of the L-nucleic acids interacting with the target molecule and mentioned in step (a). By this preferred embodiment selection may take place with regard to novel L-nucleic acid species not occurring in the starting population in a similar manner as in the biological in vivo evolution. Thus, this is a case of mirror-symmetrical in vitro evolution. Several variants of this process are conceivable, all falling under the scope of protection of the present invention.
In a preferred embodiment the invention relates to a process, whereby the interaction consists in a bond.
In a further preferred embodiment of the process of the invention the interaction consists in a catalytic reaction.
Since the molecules involved in the interaction have to interact with each other before the end of the catalytic reaction, this embodiment also implies an interaction e.g. of the (highly) affine D-nucleic acid with the optical antipode of the target molecule.
Such catalytic reactions have for example been described for ribozymes (cf. e.g. Robertson and Joyce, loc. cit.). Examples for novel catalysts have furthermore been described by Pan and Uhlenbeck (Biochemistry 33 (1994), 9561-9565), as well as by Bartel and Szostak (Science 261 (1993), 1411-1418). Lorsch and Szostak (Nature 371 (1994), 31-36) succeeded in identifying a ribozyme with kinase-activity by means of in vitro selection. Breaker and Joyce (Chem. Biol. 1 (1994), 223-229) have described a novel deoxyribozyme catalyzing the cleavage of a phosphodiester bond, while Cuenoud and Szostak (Nature 375 (1995), 611-614) were able to identify a DNA metalo-enzyme with DNA ligase activity.
In another preferred embodiment of the process of the invention the nucleic acids of step (a) are deoxyribonucleic acids.
In a particularly preferred embodiment of the process of the invention the ribonucleic acids are deoxyribozymes.
In a further preferred embodiment of the process of the invention the nucleic acids are ribonucleic acids.
In a particularly preferred embodiment of the process of the invention the ribonucleic acids are ribozymes.
This preferred embodiment of the process of the invention allows for selection of the desired molecules not by binding them to the optical antipode of the target molecule, but also by their catalytic activity. Thus, step (b) of the process of the invention possibly also comprises a cleavage of a substrate which is usually the target molecule or a part thereof, directly in connection with binding to the optical antipode of said target molecule. The cleavage of the substrate may thereby be the starting point for the further amplification and selection of suitable ribozymes. Corresponding systems have been described among others by Robertson and Joyce (loc. cit.).
In a further embodiment of the process of the invention the target molecule is an amino acid, a peptide, a polypeptide or a protein consisting of several polypeptides. The polypeptides or proteins may be glycosylated or unglycosylated.
In accordance with the invention, an amino acid, peptide, polypeptide and protein might be anyone of these (macro)molecules with which the D-nucleic acids or L-nucleic acids (?) are able to interact. Examples for such (macro)molecules are enzymes, structural proteins and hormones. Accordingly, the (macro)molecules may be components of a larger structure or be present in biological systems in diluted form.
In another preferred embodiment of the process of the invention the target molecule is a single-stranded RNA, a double-stranded RNA, a single-stranded DNA or a double-stranded DNA, as well as combinations therefrom.
It is a matter of course for the person skilled in the art that these nucleic acids may assume various conformations under various conditions. The process of the invention comprises any conformation that these (macro)molecules or their combinations might assume. The process of the invention further comprises combinations of these macromolecules. Such combinations may for example consist of single- and double-stranded RNA or DNA or of triple helices.
An antibiotic or a pharmaceutically effective substrate or its pre-stage is the target molecule of a further preferred embodiment of the process of the invention.
Preferred pharmaceutically effective substrates are e.g. steroids, ACE inhibitors, xcex2-blockers and diuretics. Thus, a therapy may effectively be interfered with by means of the L-nucleic acid of the invention and the half-life of an antibiotic or a pharmaceutically effective substrate may successfully be reduced.
In a further preferred embodiment of the process of the invention the target molecule is a sugar molecule, for example a branched or an unbranched polymeric sugar.
The term xe2x80x9csugar moleculexe2x80x9d as used herein relates to monomeric as well as to combined complex sugar structures.
In another embodiment of the process of the invention the synthesis of the L-nucleic acid of step (e) takes place in a chemical or enzymatic procedure.
In a particularly preferred embodiment of the process of the invention the chemical synthesis of the L-nucleic acid of step (e) comprises the following steps:
(ea) synthesizing L-nucleosides;
(eb) synthesizing protected L-nucleoside phosphoramidites; and
(ec) solid phase synthesis of L-nucleic acids in a synthesizer.
The invention further relates to L-nucleic acids specifically binding to a target molecule as described above.
The L-nucleic acids of the invention are preferably produced by the process of the invention. Moreover, the L-nucleic acids of the invention may be produced by any variation of the process of the invention, provided that only the selection step utilizing the optical antipode of the target molecule and the synthesizing step utilizing a D-nucleic acid as a matrix are used.
The L-nucleic acids of the invention may be used in various different ways. Due to the high affinity for the target molecule they may be used in a similar manner as monoclonal antibodies. For example, they may be provided with a marker or with a cytotoxic group. The L-nucleic acids of the invention may be used in order to stimulate or inhibit the biological function of a target molecule. Furthermore, the L-nucleic acids may be coupled to a carrier and be used as affinity material for the purifying or separating the target molecules. Immobilized L-nucleic acids may e.g. be used in order to separate enantiomers or enantiomeric impurities. Furthermore, they may be used for the purification and/or separation of cellular factors or cells. According to what is known about DNA sequences it is, e.g., possible to employ corresponding optical antipodes (D-proteins or D-peptides ) derived from the protein sequences in the process of the invention and thereby specifically produce affinity materials. Immobilized L-nucleic acids may furthermore be used as affinity material in order to separate cellular factors or cells such as in the separation of toxic components by means of dialysis.
In a particularly preferred embodiment the L-nucleic acid of the invention is L-ribonucleic acid. This L-ribonucleic acid may be present in single- or double-stranded form.
In another particularly preferred embodiment the L-nucleic acid of the invention is a ribozyme.
The present invention for the first time describes a highly affine L-oligoribonucleotide or L-oligodeoxyribonucleotide (cf. Example 1). The L-RNA identified by the process binds specifically to the D-adenosine (cf. Example 2) or L-arginine (cf. Example 3). Comparative experiments have shown that the D-form of these highly affine L-oligoribonucleotides binds the corresponding enantiomeric form of adenosine (cf. Example 2) or arginine (cf. Example 3). Thus, it could be proven that both enantiomeric forms of the highly affine oligoribonucleotide may fold up to acquire a three-dimensional structure in exactly the same manner. Thus, Pasteur""s predictions (loc. cit.) concerning the biological activity of optical enantiomers could be confirmed.
In a further particularly preferred embodiment the L-nucleic acid of the invention is an L-deoxyribonucleic acid.
As already mentioned in connection with the L-ribonucleic acids of the invention, the L-deoxyribonucleic acid may be present in single or double stranded form.
In a further particularly preferred embodiment the L-nucleic acid of the invention is a deoxyribozyme.
The invention further comprises the use of the D-nucleic acids obtained in step (c) and/or step (ca) as a matrix for the production of an L-nucleic acid with an identical nucleic acid sequence.
The invention further comprises pharmaceutical compositions containing an L-nucleic acid obtained by the above-described methods or one of the above-described L-nucleic acids, possibly in combination with a covalent modification and/or a pharmaceutically acceptable carrier.
The pharmaceutical compositions of the invention may be used in many different ways. As a matter of course, however, the effective range of the pharmaceutical composition depends on the specificity of the L-nucleic acid. The pharmaceutical compositions of the invention may for example be used in the treatment of cancer, viral and bacterial infections and high blood pressure. In every case, the physician in charge decides about the respective manner of application, dose and duration of treatment, whereby the seriousness of the disease as well as the patient""s age and general condition are some of the parameters to be considered by the physician in charge. If deemed necessary, the L-nucleic acid of the invention is dispensed in combination with a pharmaceutically acceptable carrier. The choice of additives depends, among other things, on the form of application. The person skilled in the art, however, knows from the prior art which carrier is to be added to the respective pharmaceutical composition.
Finally, the invention relates to a kit and to a diagnostic agent containing a L-nucleic acid obtained by the above-described methods or an above-described L-nucleic acid. The kit of the invention may be used for diagnostic and analytic purposes. Since, as described above, the L-nucleic acids of the invention may be used in a similar manner as antibodies, the person skilled in the art has the complete range of diagnostic possibilities of polyclonal or monoclonal antibodies at his disposal in order to use them as a field of application of the kit of the invention. The L-nucleic acid of the kit of the invention may for example be provided with a marker and for the in vitro proof of the target molecule.
L-nucleic acids may also be used as biosensors in connection with, e.g. surface-plasma-resonance-sensors, evanescent field sensors or grit coupling agents. Thus, the use of the L-nucleic acids obtained by the process of the invention or of the L-nucleic acids of the invention as biosensors are a further subject matter of the invention: it is also conceivable that the L-nucleic acids may be used as herbicides, additives in foodstuffs, for analytic methods such as determining odorous and/or taste substances or for cosmetic uses such as in anti-aging creams or sun lotions.
This and other embodiments have been disclosed to the person skilled in the art. They are obvious to her/him and comprised by the description and the examples of the present invention. For example, further literature concerning one of the above-mentioned methods, means and uses which may be applied in the sense of the present invention can be seen from the prior art, such as public libraries by using e.g. electronic auxiliary means. For this purpose, other public databases such as xe2x80x9cMedlinexe2x80x9d, to be accessed via Internet, may be consulted. Other databases and addresses are known to the person skilled in the art and may be taken from the Internet. A summary of sources and informations concerning biotechnological patents or patent applications is given in Berks, TIBTECH 12 (1994), 352-364.